[Therapeutic effect of Baimai Ointment on peripheral sensitization of chronic inflammatory pain in mice].

Chronic inflammatory pain is mainly manifested by peripheral sensitization. Baimai Ointment(BMO), a classical Tibetan medicine for external use, has good clinical efficacy in the treatment of chronic inflammatory pain, while its pharmacodynamics and mechanism for relieving peripheral sensitization remain unclear. This study established an animal model of chronic inflammatory pain induced by complete Freund's adjuvant to explore the mechanism of BMO in the treatment of chronic inflammatory pain by behavioral test, side effect assessment, network analysis, and experimental verification. The pharmacodynamics experiment showed that BMO increased the thresholds of mechanical pain sensitivity and thermal radiation pain sensitivity of chronic inflammatory pain mice in a dose-dependent manner, and had inhibitory effect on foot swelling, inflammatory mediator, and the expression of transient receptor potential vanilloid-1(TRPV1) and transient receptor potential A1(TRPA1). The results of body weight monitoring, pain sensitivity threshold detection in normal mice, rotarod performance test, and forced swimming test showed that BMO had no obvious toxic or side effect. The network analysis of 51 candidate active molecules selected according to the efficacy of BMO, content of main components, and ADME parameters showed that the inhibitory effect of BMO on chronic inflammatory pain was associated with the core regulatory elements of tumor necrosis factor(TNF) and T cell receptor signaling pathways. BMO down-regulated the protein levels of mitogen-activated protein kinase 14(MAPK14), MAPK1, and prostaglandin-endoperoxide synthase 2(PTGS2), and up-regulated the phosphorylation le-vel of glycogen synthase kinase 3 beta(GSK3 B) in the plantar tissue of mice. In conclusion, BMO can effectively relieve peripheral sensitization of chronic inflammatory pain without inducing tolerance and obvious toxic and side effects. The relevant mechanism may be related to the regulation of BMO on core regulatory elements of TNF and T cell receptor signaling pathways in surrounding tissues.

Preparation and Evaluation of Diosgenin Nanocrystals to Improve Oral Bioavailability.

Diosgenin (DSG), a well-known steroid sapogenin derived from Dioscorea nipponica Makino and Dioscorea zingiberensis Wright, has a variety of bioactivities. However, it shows low oral bioavailability due to poor aqueous solubility and strong hydrophobicity. The present study aimed to develop DSG nanocrystals to increase the dissolution and then improve the oral bioavailability and biopharmaceutical properties of DSG. DSG nanocrystals were prepared by the media milling method using a combination of pluronic F127 and sodium dodecyl sulfate as surface stabilizers. The physicochemical properties of the optimal DSG nanocrystals were characterized using their particle size distribution, morphology, differential scanning calorimetry, powder X-ray diffraction, Fourier transform infrared spectroscopy data, and solubility and dissolution test results. Pharmacokinetic studies of the DSG coarse suspension and its nanocrystals were performed in rats. The particle size and polydispersity index of DSG nanocrystals were 229.0 ± 3.7 nm and 0.163 ± 0.064, respectively. DSG retained its original crystalline state during the manufacturing process, and its chemical structure was not compromised by the nanonizing process. The dissolution rate of the freeze-dried DSG nanocrystals was significantly improved in comparison with the original DSG. The pharmacokinetic studies showed that the AUC0-72h and C max of DSG nanocrystals increased markedly (p < 0.01) in comparison with the DSG coarse suspension by about 2.55- and 2.01-fold, respectively. The use of optimized nanocrystals is a good and efficient strategy for oral administration of DSG due to the increased dissolution rate and oral bioavailability of DSG nanocrystals.

[Establishment of biological assess for quality control of Fuzi based on determination of premature ventricular contractions in rats].

Aconiti Lateralis Radix (Fuzi) is a toxic traditional Chinese medicine with definite efficacy. In order to improve the quality control of its different prepared products and ensure the security in clinic, it is significant to establish a method of quality evaluation related to clinic adverse effects. Aiming at the important biological marker of early cardiac toxicity reaction, there was no method to detect it. In this manuscript, a novel approach for measuring the minimal toxic dose (MTD) of premature ventricular contractions (PVC) poisoning of rats was established. Then, the determination methodology and conditions were optimized to meet the needs of the quality and biological assessment, including animal sex, weight, stability of standards and test solutions. Using this method, the MTD value of different Fuzi products were determined, such as Heishunpian, Baifupian, Zhengfupian, Baofupian, and Paotianxiong. The results showed that the MTD of Fuzi was significantly decreased after detoxification processed (P<0.05) and the MTD of Heishunpian, Zhengfupian, Baofupian and Baifupian was as much as 15.76, 22.36, 19.65 and 20.97 times to that of unprocessed Shengfuzi. In addition, Paotianxiong could not induce PVC in rats, which indicated that Paotianxiong was nontoxic and safe.This method could appropriately reflects the cardiotoxity of Fuzi and its prepared samples. Together with the chemical composition analysis, the contents of diester alkaloids were explored including aconitine, mesaconitine and hypaconitine as well as monoester alkaloids in Fuzi and its prepared products were significantly associated with PVC. Furthermore, there may be some components undetermined facilitating arrhythmia to be worth exploring. This research provides an overall and comprehensive approach to diagnose early clinical cardiotoxity and control the quality of Fuzi, which could not only be a complementary solution for the chemical evaluation, but a new method to ensure its efficacy and security of clinical application.

Preparation and characterisation of baicalin magnesium and its protective effect in ulcerative colitis via gut microbiota-bile acid axis modulation.

BACKGROUND: Scutellaria baicalensis Georgi is a well-known herb in traditional Chinese medicine that is frequently prescribed for various gastrointestinal conditions, including ulcerative colitis (UC). Its primary active constituent, baicalin, has poorly water solubility that reduces its efficacy. PURPOSE: To enhance the aqueous solubility of baicalin by optimising its extraction process. We compared the modulatory effects of isolated water-soluble baicalin and water-insoluble baicalin on UC, and delved deeper into the potential mechanisms of water-soluble baicalin. METHODS: We successfully extracted a more hydrophilic baicalin directly from an aqueous S. baicalensis Georgi extract through the process of recrystallisation following alcoholic precipitation of the aqueous extract obtained from S. baicalensis Georgi, eliminating the need for acid additives. This specific form of baicalin was conclusively identified by UV, IR, atomic absorption spectroscopy, elemental analysis, 1H NMR, 13C NMR, and ESI-HRMS. We subsequently compared the regulatory effects of baicalin on UC before and after optimisation, employing 16S rDNA sequencing, bile acid-targeted metabolomics, and transcriptome analysis to elucidate the potential mechanism of water-soluble baicalin; and the key genes and proteins implicated in this mechanism were verified through RT-PCR and western blotting. RESULTS: A new form of baicalin present in the aqueous solution of S. baicalensis Georgi was isolated, and its structural characterisation showed that it was bound to magnesium ions (baicalin magnesium) and exhibited favorable water solubility. Baicalin magnesium offers enhanced therapeutic benefits over baicalin for UC treatment, which alleviated the inflammatory response and oxidative stress levels while improving intestinal mucosal damage. Further investigation of the mechanism revealed that baicalin magnesium could effectively regulate bile acid metabolism and maintain intestinal microecological balance in UC mice, and suppress the activation of the nuclear factor-kappa B and peroxisome proliferator-activated receptor α signalling pathways, thereby playing a therapeutic role. CONCLUSIONS: Baicalin magnesium has good water solubility, which solves the bottleneck problem of water insolubility in the practical applications of baicalin. Moreover, baicalin magnesium exhibits therapeutic potential for UC significantly better than baicalin.

Protective effecs of baicalin magnesium on non-alcoholic steatohepatitis rats are based on inhibiting NLRP3/Caspase-1/IL-1ß signaling pathway.

Baicalin magnesium is a water-soluble compound isolated from the aqueous solution by Scutellaria baicalensis Georgi. Preliminary experiments have demonstrated that baicalin magnesium can exert protective effects against acute liver injury in rats induced by carbon tetrachloride or lipopolysaccharide combined with d-galactose by regulating lipid peroxidation and oxidative stress. The aim of this study was to investigate the protective effect of baicalin magnesium on non-alcoholic steatohepatitis (NASH) in rats and to elucidate the underlying mechanisms. NASH was induced through a high-fat diet (HFD) for 8 weeks, and Sprague-Dawley rats were intravenously injected with baicalin magnesium, baicalin, and magnesium sulfate for 2 weeks, respectively. Serum was obtained for biochemical analyses and the determination of oxidative stress indicators. Liver tissues were collected for use in liver index assessment, histopathological examination, inflammatory factor analysis, and protein and gene expression analysis. The results revealed that baicalin magnesium markedly improved HFD-induced lipid deposition, inflammatory response, oxidative stress, and histopathological impairments. And baicalin magnesium may exert a protective effect on NASH rats by inhibiting the NLR family pyrin domain involving the 3 (NLRP3)/caspase-1/interleukin (IL)-1ß inflammatory pathway. Additionally, the effect of baicalin magnesium was remarkably superior to that of equimolar baicalin and magnesium sulfate in regard to ameliorating NASH symptoms. In conclusion, the findings suggested that baicalin magnesium may represent a potential drug for the treatment of NASH.

Pharmacokinetic Analysis of Diosgenin in Rat Plasma by a UPLC-MS/MS Approach.

Diosgenin, a steroidal sapogenin, has attracted attention worldwide owing to its pharmacological properties, including antitumor, cardiovascular protective, hypolipidemic, and anti-inflammatory effects. The current diosgenin analysis methods have the disadvantages of long analysis time and low sensitivity. The aim of the present study was to establish an efficient, sensitive ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach for pharmacokinetic analysis of diosgenin amorphous solid dispersion (ASD) using tanshinone IIA as an internal standard (IS). Male Sprague-Dawley rats were orally administered diosgenin ASD, and orbital blood samples were collected for analysis. Protein precipitation was performed with methanol-acetonitrile (50 : 50, v/v), and the analytes were separated under isocratic elution by applying acetonitrile and 0.03% formic acid aqueous solution at a ratio of 80 : 20 as the mobile phase. MS with positive electron spray ionization in multiple reaction monitoring modes was applied to determine diosgenin and IS with m/z 415.2⟶271.2 and m/z 295.2⟶277.1, respectively. This approach showed a low limit of quantification of 0.5 ng/ml for diosgenin and could detect this molecule at a concentration range of 0.5 to 1,500 ng/ml (r = 0.99725). The approach was found to have intra- and inter-day precision values ranging from 1.42% to 6.91% and from 1.25% to 3.68%, respectively. Additionally, the method showed an accuracy of -6.54 to 4.71%. The recoveries of diosgenin and tanshinone IIA were 85.81-100.27% and 98.29%, respectively, with negligible matrix effects. Diosgenin and IS were stable under multiple storage conditions. Pharmacokinetic analysis showed that the C max and AUC0⟶t of diosgenin ASD were significantly higher than those of the bulk drug. A sensitive, simple, UPLC-MS/MS analysis approach was established and used for the pharmacokinetic analysis of diosgenin ASD in rats after oral administration.

Baicalin Magnesium Salt Attenuates Lipopolysaccharide-Induced Acute Lung Injury via Inhibiting of TLR4/NF-κB Signaling Pathway.

Baicalin (BA) magnesium salt (BA-Mg) is a good water-soluble ingredient extracted from Scutellaria baicalensis Georgi, a commonly used traditional Chinese medicine. This study is aimed at investigating whether BA-Mg could exert a better protective effect on lipopolysaccharide- (LPS-) induced acute lung injury (ALI) in mice and illuminate the underlying mechanisms in vivo and in vitro. Mice were intraperitoneally administrated with equimolar BA-Mg, BA, and MgSO4 before LPS inducing ALI. Lung tissues and bronchoalveolar lavage fluid were collected for lung wet/dry ratio, histological examinations, cell counts, and biochemical analyses at 48 h post-LPS exposure. Meanwhile, the protein expressions of TLR4/NF-κB signaling pathway and proinflammatory cytokines in lung tissues and lung bronchial epithelial cells (BEAS-2B) were detected. The results showed BA-Mg pronouncedly ameliorated LPS-induced inflammatory response and histopathological damages, elevated antioxidant enzyme activity (SOD), and downregulated myeloperoxidase (MPO) and malonaldehyde (MDA) levels through the inhibition of TLR4/NF-κB signaling pathway activation. Moreover, the effect of BA-Mg was significantly better than that of BA and MgSO4 in ameliorating symptoms. Overall, BA-Mg can effectively relieve inflammatory response and oxidative stress triggered by LPS, indicating it may be a potential therapeutic candidate for treating ALI.

Soluplus-Mediated Diosgenin Amorphous Solid Dispersion with High Solubility and High Stability: Development, Characterization and Oral Bioavailability.

BACKGROUND AND PURPOSE: The traditional Chinese medicine, diosgenin (Dio), has attracted increasing attention because it possesses various therapeutic effects, including anti-tumor, anti-infective and anti-allergic properties. However, the commercial application of Dio is limited by its extremely low aqueous solubility and inferior bioavailability in vivo. Soluplus, a novel excipient, has great solubilization and capacity of crystallization inhibition. The purpose of this study was to prepare Soluplus-mediated Dio amorphous solid dispersions (ASDs) to improve its solubility, bioavailability and stability. METHODS: The crystallization inhibition studies were firstly carried out to select excipients using a solvent shift method. According to solubility and dissolution results, the preparation methods and the ratios of drug to excipient were further optimized. The interaction between Dio and Soluplus was characterized by differential scanning calorimetry (DSC), fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), powder X-ray diffraction (PXRD) and molecular docking. The pharmacokinetic study was conducted to explore the potential of Dio ASDs for oral administration. Furthermore, the long-term stability of Dio ASDs was also investigated. RESULTS: Soluplus was preliminarily selected from various excipients because of its potential to improve solubility and stability. The optimized ASDs significantly improved the aqueous solubility of Dio due to its amorphization and the molecular interactions between Dio and Soluplus, as evidenced by dissolution test in vitro, DSC, FT-IR spectroscopy, SEM, PXRD and molecular docking technique. Furthermore, pharmacokinetic studies in rats revealed that the bioavailability of Dio from ASDs was improved about 5 times. In addition, Dio ASDs were stable when stored at 40°C and 75% humidity for 6 months. CONCLUSION: These results indicated that Dio ASDs, with its high solubility, high bioavailability and high stability, would open a promising way in pharmaceutical applications.

Design and evaluation of San-huang dispersible tablet--an efficient delivery system for Traditional Chinese Medicine.

San-huang dispersible tablet (SHDT) was designed with a patented technology to enrich the active ingredients in rhubarb and with a wide selection of excipients in the new manufacturing procedure. The total rhubarb anthraquinones were first enriched in the extract by our patented technology. Eudragit L100, S100 and PEG-6000 were used to release a part of the total rhubarb anthraquinones at the colon to induce the cathartic effect of the anthraquinones by another patented technology. Microcrystalline Cellulose (MCC), low-substituted hydroxypropyl cellulose (L-HPC), sodium carboxymethyl starch (CMS-Na), and hydroxypropyl methylcellulose (HPMC) were used to ensure quick release of baicalin and berberine hydrochloride in the stomach. The dissolution of SHDT was evaluated by a method in 2005 Chinese Pharmacopoeia along with San-huang tablet (SHT), and the results demonstrated that the dissolution of baicalin and berberine hydrochloride more than double that of SHT and release of half of the rhubarb anthraquinones in colon.

A new sensitive UPLC-MS/MS method for the determination of cucurbitacin B in rat plasma: application to an absolute bioavailability study.

Cucurbitacin B (CuB) is a highly oxygenated tetracyclic triterpene, and a Biopharmaceutics Classification System (BCS) class IV drug used for the treatment of persistent hepatitis, chronic hepatitis, and primary liver cancer. Nevertheless, CuB has low solubility and low permeability, and is present at low concentrations in the human body. The aim of this study was to develop a method for the determination of CuB in plasma using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) with estrone as an internal standard (IS), as well as to examine the pharmacokinetics and absolute bioavailability of CuB in rats. Plasma samples were processed by liquid-liquid extraction with ethyl acetate. Separation was achieved on a BEH C18 column (2.1 × 50 mm, 1.7 µm) at 35 °C using an isocratic mobile phase system with 0.1% formic acid-acetonitrile (50 : 50, v/v) at a flow rate of 0.3 mL min-1. The detection was performed using a multiple reaction monitoring mode via a positive electrospray ionization interface. The calibration curves showed good linearity (r = 0.9998) within the tested concentration ranges. The lower limit of quantification for plasma was 0.05 ng mL-1; the matrix effect of CuB and IS was 94.19-99.42% and 100.83%, respectively. The mean extraction recoveries from plasma were 85.34-90.53%. The intra-day and inter-day accuracies and precision deviations were within ±15%, which was in line with the allowable range of accuracy. In addition, the stability of the method was also verified. The absolute bioavailability of orally administered CuB in rats was 1.37%. To sum up, the presented method was determined to be suitable for the quantitation of CuB in rat plasma. Also, the absolute bioavailability observed in the present study suggested that it was necessary to change the dosage form to improve bioavailability, or to improve this by other means.

[Sanhuang tablets research].

Sanhuang tablets is one of common traditional Chinese patent preparation, it has effects of clear fever, detoxifcation, dispel inflammation, purgation. It was contained in the ministerial standards of Ministry of Health in 1997, and was contained in Chinese pharmacopoeia version 1 of 2000 and 2005. Its improvement of dosage form, preparation technique, quality analysis, pharmacology and clinical usage were reviewed in this paper.

Oral colon-specific drug delivery system reduces the nephrotoxicity of rhubarb anthraquinones when they produce purgative efficacy.

Rhubarb is commonly used to treat constipation in China and anthraquinones (AQs) are the active components present in rhubarb. However, an increasing number of studies have reported that AQs induce nephrotoxicity. In the present study, rhubarb total free anthraquinones (RTFA) oral colon-specific drug delivery granules (RTFA-OCDD-GN) were prepared to determine whether RTFA-OCDD-GN could reduce the nephrotoxicity that occurs when AQs produce purgative efficacy. RTFA-OCDD-GN were prepared using pH-enzyme double-layer coating technology and the cumulative release rate of RTFA in RTFA-OCDD-GN was assessed. The first black stool time, the number and state of feces over 8 h were observed to measure the purgative efficacy. In the nephrotoxicity test, biochemical and histopathological examinations were performed following 20 and 40 days administration, and 20 days convalescence. The cumulative release rate of RTFA in RTFA-OCDD-GN was >80% in simulated colonic fluid. RTFA-OCDD-GN produced considerable purgative efficacy compared with rhubarb medical material samples (RMMS). Following 40 days RMMS administration, blood urea nitrogen, creatinine and urine ß2-microglobulin levels in the high-dosage group were significantly increased compared with the control and RTFA-OCDD-GN groups (P<0.05). All specimens from the high-dosage RMMS group exhibited swelling/degeneration of renal proximal convoluted tubule epithelial cells. No difference in pathological conditions and biochemical indicators was detected between the RTFA-OCDD-GN groups and the control group. The nephrotoxicity of AQs was significantly reduced following RTFA-OCDD-GN administration, which produced considerable purgative efficacy compared with RMMS.

The pharmacokinetic study on the mechanism of toxicity attenuation of rhubarb total free anthraquinone oral colon-specific drug delivery system.

Rhubarb is commonly used as laxatives in Asian countries, of which anthraquinones are the major active ingredients, but there are an increased number of concerns regarding the nephrotoxicity of anthraquinones. In this study, we compared the pharmacokinetic characteristics of rhubarb anthraquinones in rats after orally administered with rhubarb and rhubarb total free anthraquinone oral colon-specific drug delivery granules (RTFA-OCDD-GN), and then explained why these granules could reduce the nephrotoxicity of anthraquinones when they produced purgative efficacy. A sensitive and reliable high performance liquid chromatography (HPLC) method has been fully validated for simultaneous determination of the five active components of rhubarb, and successfully applied to investigate and compare the remarkable differences in pharmacokinetic study of rhubarb anthraquinones after orally administered with rhubarb and RTFA-OCDD-GN. The results showed that, compared with rhubarb group, the AUC, Cmax, t1/2z and Vz/F of aloe-emodin, rhein, emodin and chrysophanol in rats receiving the RTFA-OCDD-GN were significantly decreased, and the Tmax of the four analytes was prolonged. Moreover, the Tmax of rhein, the Cmax of chrysophanol and emodin all have significant differences (P<0.05). Simultaneously, anthraquinone prototype excretion rates in urine and feces of aloe-emodin, rhein, emodin, chrysophanol and physcion were all increased. These findings suggested that oral colon-specific drug delivery technology made anthraquinone aglycone to colon-specific release after oral administration. This allowed anthraquinones to not only play the corresponding purgative effect but also avoid intestinal absorption and promote excretion. And thereby greatly reduced the nephrotoxicity of rhubarb. The result is a new breakthrough in rhubarb toxicity attenuated research.

Design and evaluation of rhubarb total free anthraquinones oral colon-specific drug delivery granules to improve the purgative effect

Rhubarb is commonly used as a cathartic in Asian countries. However, researchers have devotedextensive concerns to the quality control and safety of rhubarb and traditional Chinese preparations composed of rhubarb due to the instable purgative effect and potential nephrotoxicity of anthraquinones. In this study, we aimed to prepare rhubarb total free anthraquinones (RTFA) oral colon-specific drug delivery granules (RTFA-OCDD-GN) to delivery anthraquinones to colon to produce purgative effect. RTFA-OCDD-GN were prepared using chitosan and Eudragit S100 through a double-layer coating process and the formulation was optimized. Continuous release studies were performed in a simulated gastric fluid (pH 1.2), followed by a small-intestinal fluid (pH 6.8) and a colonic fluid (pH 7.4, containing rat cecal contents). The purgative effect test was performed in rats. The dissolution profile of RTFA-OCDD-GN showed that the accumulative dissolution rate of RTFA was about 83.0% in the simulated colonic fluid containing rat cecal contents and only about 9.0% in the simulated gastrointestinal fluids. And the RTFA-OCDD-GN could produce the comparative purgative activity as rhubarb, suggesting it could deliver the free AQs to the colon. The RTFA-OCDD-GN was a useful media to enhance the purgative activity of free anthraquinones after administered orally.